Lignocellulosic biomass represents one of the most abundant renewable resources on Earth. It is formed of three major components: cellulose, hemicellulose, and lignin, and includes, for example, agricultural and forestry residues, municipal solid waste (MSW), fiber resulting from grain operations, waste cellulosic products (e.g., paper and pulp operations), and energy crops. The cellulosic and hemicellulosic polymers of biomass can be hydrolyzed into their component sugars, such as glucose and xylose, which can then be fermented by microorganisms to produce ethanol. Conversion of even a small portion of the available biomass into ethanol could substantially reduce current gasoline consumption and dependence on petroleum.
Many conversion processes are known that breakdown lignocellulosic biomass to produce bioenergy. These processes vary from multi-enzyme and multi-fermentation approaches called separate hydrolysis and fermentation (SHF) [Wilke et al. (1976) Biotechnol. Bioeng. Symp. 6:55] to simpler, simultaneous cellulose hydrolysis (or saccharification) and fermentation (SSF) [Takagi et al. (1977) in Proceedings of the Bioconversion Symposium, Indian Institute of Technology, New Delhi, pp. 55-571; Spindler (1988) Appl. Biochem. Biotechno1.17:279-294; Alfani (2000) J. Ind. Microbiol. Biotechnol. 25:184-192]. In an SHF process, the cellulosic biomass is hydrolyzed with cellulases to liberate fermentable glucose followed by a separate step for fermentation to ethanol. The SSF process combines the enzymatic hydrolysis and fermentation simultaneously, reducing the process complexity. A natural extension is simultaneous saccharification and cofermentation (SSCF) using microorganisms that are able to convert both hexose and pentose sugars to ethanol. This process simplification culminates with the development of fermentation microorganisms that produce their own enzymes for cellulose hydrolysis, called consolidated bioprocessing (CBP). CBP involves four biologically-mediated events: (1) enzyme production, (2) substrate hydrolysis, (3) hexose fermentation and (4) pentose fermentation. In contrast to the other approaches, where some or all of the steps may be performed independently, all four events are performed simultaneously in a CBP configuration.
While chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity, it also produces microbial growth inhibitors such as furan and phenolic aldehydes [Klinke et al. (2004) Appl. Microbiol. Biotechnol. 66: 10-26]. The most abundant inhibitors, 5-hydroxymethyl furfural (5-HMF) and furfural, are generated from the dehydration of glucose and xylose, respectively, under acidic pH at high temperatures. These aldehydes impart broad cytological and physiological damage, especially in ethanologenic fungi and bacteria [Taylor et al. (2012) Biotechnol. J. 7:1169-1181; Palmqvist et al. (2000a) Bioresource Technol. 74:17-24]. Hence, for processes which involve fermentation, there is a need to abate the microbial inhibition that can arise during biomass pretreatment.
Several non-biological strategies have been described in the literature for removal of pretreatment inhibitors from lignocellulosic hydrolysates, including overliming with Ca(OH)2 or NaOH to precipitate inhibitors and addition of activated charcoal or anion exchange resins to adsorb toxic compounds [Taylor 2012; Palmqvist et al. (2000b) Bioresource Technol. 74:25-33]. Biological abatement has been evaluated either by adding enzymes to hydrolysates to degrade compounds (generally specific for phenolic, lignin-derived inhibitors) or by adding microorganisms capable of directly metabolizing pretreatment inhibitors. For example, Li et al. showed that Cupriavidus necator can rapidly reduce furfural to the less toxic form, furfuryl alcohol [Li et al. (2011a) Biodegradation 22:1215-1225]. Conceptually, this microorganism could be applied to pretreatment hydrolysates to scavenge furan aldehydes; however, the microorganism requires oxygen for growth and does not grow at elevated temperatures, whereas many industrial processes are conducted at elevated temperatures under anaerobic conditions, making this microorganism unsuitable for such processes.
To improve inhibitor tolerance, fermentative, biofuel-producing microorganisms have been adapted or genetically modified to provide robust growth and performance in the presence of pretreatment hydrolysates. For example, improved inhibitor tolerance has been engineered into common ethanologenic microorganisms, including Saccharomyces cerevisiae [Almeida et al. (2007) J. Chem. Technol. Biotechnol. 82:340-349; Larsson et al. (2001) Appl. Environ. Microbiol. 67:1163-1170], Zymomonas mobilis [Yang et al. (2010) Bmc Microbiol. 10:135)], and ethanologenic Escherichia coli [Wang et al. (2011) Appl. Environ. Microbiol. 77:5132-5140; Wang et al. (2012) Appl. Environ. Microbiol. 78:2452-2455; Zheng et al. (2012) Appl. Environ. Microbiol. 78:4346-4352]. Further, enzymatic detoxification of furan aldehydes has been widely documented in yeast [Liu et al. (2004) J. Ind. Microbiol. Biotechnol. 31:345-352; Bowman et al. (2010) Appl. Environ. Microbiol. 76:4926-4932; Park et al. (2011) Bioresource Biotechnol. 102:6033-6038] and in E. coli [Miller et al. (2009) Appl. Environ. Microbiol. 75:4315-4323; Wang et al. (2011)], which generally include aldehyde-specific oxidoreductases or alcohol dehydrogenases. While these microorganisms have been important for first-generation ethanol production, they are not suitable for second generation biofuels which use thermophilic, cellulolytic strains that can directly solubilize cellulose and ferment carbohydrates into fuels under anaerobic conditions [Elkins et al. (2010) Curr. Opin. Biotechnol. 21:657-662; Lynd, L. R. et al. (2008) Nat. Biotechnol. 26:169-172; Olson et al. (2012) Curr. Opin. Biotechnol. 23:396-405)].
Hence a need remains for anaerobic, inhibitor-tolerant microorganisms capable of fermentation at elevated growth temperatures (typically above 50-60° C., and even as high as 80° C.). To address this need, the saccharolytic thermophile Thermoanaerobacter pseudethanolicus 39E (Teth39E) was grown in the presence and absence of furfural and a protein that was up-regulated 7-fold was selected for further study. From the genomic sequence of Teth39E, this protein was identified as the product of open reading frame (orf) Teth39_1597 (hereinafter referred to as “the bdhA gene” or “bdhA”) and found to encode an iron-dependent alcohol dehydrogenase (hereinafter referred to as “BdhA”).
Alcohol dehydrogenases (ADHs) constitute a large family of enzymes and catalyze the reversible oxidation of primary or secondary alcohols to aldehydes or ketones. In bacteria and yeast, ADHs (also referred to herein and in the literature as aldehyde reductases) have been found that are capable of reducing furfural and 5-(hydroxymethyl) furfural (5-HMF) (and other toxic aldehydes) to alcohols. However, those enzymes which have heretofore been studied are distinct from that encoded by the bdhA gene.
For example, S. cerevisiae has multiple NADH and NADPH-dependent aldehyde reductases that can convert furfural and 5-HMF to non-toxic alcohols [Liu et al. (2008) Appl. Environ. Microbiol. 81:743-753]. The S. cerevisiae ADH6 gene product has been characterized as a Zn- and NADPH-dependent enzyme capable of reducing 5-HMF (Larroy et al. (2002) Biochem. J. 361:163-172; Petersson et al. (2006) Yeast 23:455-464). U.S. Patent Appln. Pub. No. 2007/0155000 also describes ethanol-producing S. cerevisiae strains that tolerate furfural and 5-HMF by overexpressing the yeast ADH6 gene. U.S. Pat. No. 8,110,387 describes S. cerevisiae ADH1 and mutants thereof that have NADH-dependent 5-HMF reductase activity and indicates that these enzymes can aid in detoxifying lignocellulosic hydrolysates. U.S. Pat. No. 7,253,001 relates to S. cerevisiae strains with improved xylose utilization which were created by deleting an endogenous aldehyde dehydrogenase gene and introducing 5 other genes. U.S. Patent Appln. Pub. No. 2012/0190089 describes recombinant yeast with engineered metabolic pathways to produce isobutanol, in part by expressing an exogenous NADH-dependent ADH that converts isobutyraldehyde to isobutanol under anaerobic conditions. None of these yeast genes are homologous to BdhA.
U.S. Pat. No. 8,039,239 describes recombinant Clostridia strains that overexpress an NADPH-dependent secondary alcohol dehydrogenase with sequence homology to a previously-characterized T. pseudethanolicus NADPH-dependent alcohol dehydrogenase (Teth39 _0218). However, as shown in FIG. 4 of the '239 patent, the amino acid sequence of that ADH is markedly distinct from BdhA.
ADHs distinct from BdhA also exist in other bacteria. For example, U.S. Patent Appln. Pub. No. 2011/0177579 describes a thermostable, primary-secondary ADH from Thermococcus guaymasensis which appears related to Zn-dependent ADHs and it is unknown whether the enzyme is capable of detoxifying furfural or 5-HMF. U.S. Patent Appln. Pub. No. 2012/0108855 reports recombinantly-produced, ethanologenic bacteria with increased expression of the transhydrogenase genes pntA and pntB which are capable of imparting increased furfural tolerance. The C. necator strain mentioned above turns out to have a NADH- and Zn-dependent ADH [Li et al. (2011b) Biodegradation 22:1227-1237].
In accordance with the invention, the discovery of BdhA provides a route to modified anaerobic, aldehyde-tolerant thermophilic microorganisms suitable for use in bioprocessing lignocellulosic biomass to efficiently produce biofuel.